A third of the world's population is infected with Mycobacterium tuberculosis (Mtb), and most of these infections are latent. Tubercle bacilli can remain inactive in lung lesions only to emerge decades later to seed new outbreaks of tuberculosis. In addition, tuberculosis is one of the most difficult bacterial infections to treat and continues to cause more deaths than any other bacterial infection. Bacilli exist in replicating and non-replicating states in a range of microenvironments that vary in oxygen concentration and nutrient availability. The bacilli that survive during latent infection likely exist in a non-replicating state and antimicrobials, effective against actively growing bacteria, are often not effective against non-replicating bacteria. Little is known about the metabolic mechanisms employed by Mtb to survive latent infection or the hostile microenvironment of necrotic caseous tubercle lesions where oxygen is limited and nutrient sources suboptimal. Mtb cannot grow but endures in the absence of aerobic respiration. Therefore, the non-replicating anaerobic state is considered a prime model for persistent bacilli in vivo. In the absence of aerobic respiration Mtb requires a functional electron transport system to drive ATP synthesis. However, the core metabolic mechanisms by which the bacilli maintain redox balance are unknown. Therefore, our central research question is "What metabolic mechanisms are employed by Mtb when aerobic respiration is inhibited?" At least three factors limit Mtb aerobic respiration: the inhibitory effect of macrophage-produced nitric oxide and carbon monoxide, and the structure of mature granulomas. All three of these conditions strongly induce the DosR regulon, a regulon essential for anaerobic survival. However, the regulon does not encode a complete recognizable intermediary metabolic pathway. Our expression analysis and biochemical data strongly indicate that Mtb - an obligate aerobe - maintains a unique multifaceted intermediary pathway for metabolism in the absence of aerobic respiration. Thus, throughout the evolution of Mtb as a frank human pathogen, it has maintained an extensive array of enzymes which appear to be geared specifically for anaerobic metabolic functions. The pathway predicts that Mtb has the potential to metabolize lipids and all other major carbon sources anaerobically. Antimicrobials designed to kill non-replicating anaerobic bacilli are sorely needed. Our research directly supports this goal. New antimycobacterial drugs and drug combinations are routinely tested against hypoxic/anaerobic bacilli, but questions about the proper protocol and our limited understanding of relevant intermediary metabolic pathways limit a rational approach to drug design. Our preliminary data suggests we are at the brink of a fundamental understanding of Mtb anaerobic metabolism. Our working hypothesis is: Mtb employs a novel anaerobic metabolic cycle in conjunction with the DosR regulon to confer survival during non- respiring conditions within TB lesions. To test this hypothesis we will investigate key aspects of the proposed anaerobic metabolic pathway. PUBLIC HEALTH RELEVANCE: A third of the world's population is latently infected with Mycobacterium tuberculosis. Tuberculosis is one of the most difficult bacterial infections to treat and continues to cause more deaths than any other bacterial infection. The bacteria that persist during latent infection and antibacterial treatment are likely in non-replicating states with low levels of metabolic activity. Our research is designed to demonstrate the unique metabolic pathway used by M. tuberculosis to survive in a non-replicating state in order to provide mechanisms that can be targeted by novel antimicrobials.